Chicken RNA polymerase I promoter and the use thereof

p-0002The present invention relates to a novel polynucleotide that possesses a transcription-promoting activity, to vectors containing this polynucleotide, and to the use thereof for the transcription of sequences of interest, such as for the production of uncapped viral RNA. The present invention also relates to host cells, preferably of avian origin, that contain a polynucleotide or a vector in accordance with the invention.

p-0003The prevention of influenza rests primarily on vaccination, which is strongly recommended. In France, for example, 100% of the cost of vaccination is covered by the French national health insurance agency for persons at risk, which are primarily those subjects 65 years of age and older and subjects suffering from chronic respiratory, cardiovascular, renal, and metabolic afflictions. Post-vaccine immunity is conferred by the production of antibodies directed against surface glycoproteins, in particular against hemagglutinin (HA) but also against neuraminidase (NA).

h-0001I—Current Methods: Production Using Fertilized Chicken Eggs and Inactivation

p-0004Influenza vaccines are composed of three different virus strains: two Type A (H3N2 and H1N1) and a Type B. The choice of vaccine strains is reevaluated each year in accordance with the monitoring data for the variations in circulation, and is the subject of a WHO recommendation which is announced in mid-February for the northern hemisphere. The vaccines used today, as they have been for the last thirty years, are composed of viruses reproduced in fertilized chicken eggs and then inactivated (Manuguerra, 2001, <i>Repères sur les infections bronchopulmonaires</i>, pp. 328-342. Edited by P. Léophonte &amp; Y. Mouton: PIL).

p-0005For Type A viruses, reassortant viruses with high reproduction capacities in fertilized eggs and with the HA and NA of the variations indicated by the WHO are prepared and provided to the industrial vaccine producers. One or two fertilized eggs are required to manufacture a dose of trivalent vaccine. Each vaccine manufacturer performs the purification step differently, but all follow the same principle: allantoic fluid is taken and the viruses are inactivated by treatment with formaldehyde or β-propiolactone before being purified by ultracentrifugation. The viruses may be fragmented using lipid and/or detergent solvents such as Tween 80. The whole or fragmented viruses are then adjusted to the standard level of 15 μg of HA per dose of vaccine, per strain. A purification step in the presence of dialyzable detergent makes it possible to produce a subunit vaccine that contains nothing more than the envelope glycoproteins (HA and NA) of the virus. Because of the stronger reactogenicity of vaccines that contain whole viruses, it is in fact the fragmented and subunit vaccines which are marketed today. The period necessary for a vaccine to be produced and quality tested in conformity with European standards before its release on the market is about 6 months.

p-0006Comprised of inactivated viruses, influenza vaccines are generally very well tolerated and cause only minor adverse reactions, such as local reactions at the point of injection and febrile reactions and cephalgias in the 48 hours following injection. The contraindications for influenza vaccination are limited to allergies to egg proteins, primarily egg albumin, and to allergies to manufacturing residues or mercurothiolate. In the absence of information on the effects which it may have on fetal development, influenza vaccination is not recommended for women in the first trimester of pregnancy.

p-0007The protective effectiveness of a vaccine comprised of inactivated viruses against a subsequent influenza infection is difficult to evaluate because the effectiveness may vary widely from one season to another, in particular as a function of the degree of the antigenic relationship between the circulating viruses and those included in the vaccine, and because effectiveness also depends on the state of immunization of the vaccinated person. In France, the improvement in vaccine coverage between 1979 and 1999 was accompanied by a reduction in influenza-related mortality (approximately 20,000 deaths per year in 1979; 2,500 deaths per year since 1985), which suggests, if not demonstrates, a positive impact by vaccine policy. In addition, evidence from a number of studies makes it possible to estimate that vaccinations for the elderly can decrease the number of viral pneumonias and hospitalizations by half and the number of fatal influenza cases by about seventy percent (Large et al., 1995, Annals of Internal Medicine, 123, 518-527).

h-0002II—Developmental Prospects for Influenza Vaccines

p-0008Several avenues of research are currently under investigation for the purpose of 1) improving the intensity, quality, and duration of responses induced by influenza vaccines, and enlarging their specificities; and 2) allowing a rapid response should a virus of a potentially pandemic sub-type emerge. Work concerning the production of live vaccines as well as the production of vaccine viruses in cell cultures are summarized below. Among the other avenues of research explored, the use of adjuvants, recombinant subunit vaccines, and polynucleotide vaccines should also be noted.

h-0003—Production of Live Vaccines

p-0009A live influenza vaccine is likely to induce a broader and longer-lasting immunity because, in particular, it stimulates immunity via cellular mediation. A live vaccine is also likely to be better accepted by the general population because it is administered by the nasal, and not parenteral, route. Two types of live vaccines can be envisaged today: live attenuated vaccines and live recombinant vaccines.

p-0010Live Attenuated Vaccines

p-0011Attenuated strains are obtained by the reassortment of circulating wild strains with a donor strain that has been attenuated by cold adaptation and whose genotype is well characterized, however without perfect establishment of the relationships between the mutations observed and the phenotype (attenuation, cold adaptation, heat-sensitivity) (Keitel et al., 1998, Textbook of Influenza, pp. 373-390. Edited by K. G. Nicholson, R. G. Webster &amp; A. J. Hay. Oxford, England: Blackwell Science, Ltd.).

p-0012The method for producing attenuated vaccines in fertilized chicken eggs is analogous to that described above, but without the virus inactivation step. The results of clinical tests conducted recently by American researchers indicate that trivalent vaccines comprised of attenuated strains administered by intranasal route are harmless for adults, the elderly, and children, with only throat irritation and nasal discharge during the 48 hours following vaccination as side effects. They may have greater protective efficacy than inactivated vaccines, particularly due to the induction of an IgA antibody mucosal response, but their transmission and reversion potentials remain to be documented. Vaccination with live attenuated viruses has been practiced widely in Russia for several decades, but it is difficult to draw convincing overall conclusions because of the variety of conditions employed.

p-0013Live Recombinant Vaccines

p-0014The recent development of reverse genetics systems for influenza viruses (Neumann et al., 2001, Virology 287, 243-50) makes it possible to envisage the production of recombinant viruses in the genome with genetic modifications specifically introduced in order to confer properties of adequate attenuation and immunogenicity. This approach offers increased security (because of the absence of contaminating agents such as those which may introduced when viral strains are sampled and the possibility of more easily minimizing and controlling the risks-of reversion of the vaccine virus) and would make it possible to respond more specifically to varied epidemic situations. The reverse genetics systems in existence today are based on the use of the human RNA polymerase I transcription system. Because of the narrow species specificity of RNA polymerase I, its use is restricted to cells of human or primate origin. It can be used for the production of recombinant vaccine viruses in cell cultures but not in fertilized chicken eggs.

h-0004—Production of Vaccine Viruses in Cell Cultures

p-0015The principal advantages of using fertilized chicken eggs in the production of vaccine influenza viruses are: i) a greatly increased virus yield; and ii) a minimum risk of contamination by agents pathogenic to man, a particular consideration in the production of non-inactivated vaccines. The possibility of producing vaccine viruses in cells cultivated in a medium lacking serum is also explored. The companies Baxter and Solvay recently approved banks of cells derived from the Vero and MDCK lines, respectively, for the production of vaccine viruses. A vaccine produced in MDCK cells and inactivated received marketing authorization in 2001 but has yet to be commercialized.

p-0016Several types of influenza vaccines, both inactivated and live, and both those produced in fertilized chicken eggs and those produced in cell cultures, will likely coexist in the future marketplace. Among these some may appear particularly indicated for the vaccination of a certain category of subjects, and less indicated for the vaccination of another category. In the event of a pandemic, all production means available, using fertilized eggs and cell cultures, will need to be mobilized.

p-0017The present invention responds to these needs and other needs as will be apparent to the skilled reader of the present description of the invention.

p-0018The present invention relates to a polynucleotide, to recombinant DNA that contain the aforesaid polynucleotide, and to the use thereof, preferably for the production of uncapped RNA viruses to be used in a vaccine.

p-0019More specifically, the present invention has as an aim an isolated or purified polynucleotide which includes sequence SEQ ID NO: 1, presented in <figref idrefs="DRAWINGS">FIG. 1</figref>, or a fragment thereof, which preferably has a transcription-promoting activity.

p-0020The present invention also relates to an isolated or purified polynucleotide that includes sequence SEQ ID NO: 2, presented in <figref idrefs="DRAWINGS">FIG. 2</figref>, or a fragment thereof, which preferably has a transcription-promoting activity.

p-0021The invention also relates to: a recombinant DNA that includes one of the polynucleotides in accordance with the invention, or a fragment thereof; a vector that includes one of the polynucleotides in accordance with the invention, or a recombinant DNA, or a fragment of recombinant DNA; a host cell and a fertilized egg of avian origin that contains a polynucleotide and/or a recombinant DNA in accordance with the invention.

p-0022The invention also relates to the use of at least one of the following elements for the production of a sequence of interest such as, for example, an uncapped recombinant RNA virus: <ul><li id="ul0001-0001" num="0000"><ul><li id="ul0002-0001" num="0022">a polynucleotide in accordance with the invention;</li><li id="ul0002-0002" num="0023">a recombinant DNA in accordance with the invention;</li><li id="ul0002-0003" num="0024">a vector in accordance with the invention;</li><li id="ul0002-0004" num="0025">a host cell in accordance with the invention; and</li><li id="ul0002-0005" num="0026">a fertilized egg of avian origin in accordance with the invention.</li></ul></li></ul>

p-0023In a specific embodiment, the invention relates to a method for the production of uncapped recombinant viruses by reverse genetics, wherein the method comprises the following steps: <ul><li id="ul0003-0001" num="0000"><ul><li id="ul0004-0001" num="0028">a) introducing one or several vectors into a cell or fertilized egg of avian origin, the aforesaid vector or vectors comprising a recombinant DNA in accordance with the invention, and the DNA that express the proteins necessary for the transcription/replication of viral RNA;</li><li id="ul0004-0002" num="0029">b) applying to the cell or egg obtained in a) conditions that make possible the expression of recombinant viruses; and</li><li id="ul0004-0003" num="0030">c) recovering the recombinant viruses obtained in b).</li></ul></li></ul>

p-0024In a preferred embodiment, the present invention proposes a method for the production of recombinant influenza viruses by reverse genetics, wherein the method comprises the following steps: <ul><li id="ul0005-0001" num="0000"><ul><li id="ul0006-0001" num="0032">a) introducing into an avian cell or fertilized chicken egg at least one vector that comprises a recombinant DNA provided by a preferred method of the invention, and vectors that express the PA, PB1, PB2, and NP proteins of an influenza virus;</li><li id="ul0006-0002" num="0033">b) applying to the cell or egg obtained in a) conditions that make possible the expression of recombinant influenza viruses; and</li><li id="ul0006-0003" num="0034">c) recovering the recombinant influenza viruses obtained in b).</li></ul></li></ul>

p-0025An aim of the invention is a compound that includes a pharmaceutically acceptable vehicle and a recombinant virus produced by a method in accordance with the present invention.

p-0026A particular aim of the, invention is an anti-influenza compound that includes a pharmaceutically acceptable vehicle and a recombinant influenza virus produced by a production method in accordance with the present invention.

p-0027The present invention also relates to a therapeutic compound that includes a sequence transcribed by a polynucleotide in accordance with the invention.

p-0028Advantageously, cloning the chicken RNA polymerase I promoter makes it possible to develop a reverse genetics system adapted to the production of recombinant vaccine viruses in fertilized chicken eggs, a system which may prove particularly well adapted to the production of live vaccines.

BRIEF DESCRIPTION OF FIGURES

p-0029<figref idrefs="DRAWINGS">FIG. 1</figref> shows a sequence (SEQ ID NO: 1) that corresponds to the AgeI-SacI chicken genomic DNA fragment which contains the chicken RNA polymerase I promoter.

p-0030<figref idrefs="DRAWINGS">FIG. 2</figref> shows a second fragment of chicken genomic DNA (BsaI-SacI) of sequence SEQ ID NO: 2 which contains the chicken RNA polymerase I promoter.

p-0031<figref idrefs="DRAWINGS">FIG. 3</figref> shows the protocol used to analyze the efficiency of transcription of the CAT sequence using the sequences derived from C13-18 cosmid.

p-0032<figref idrefs="DRAWINGS">FIG. 4</figref> shows the results of the iterative analysis of the restriction fragments derived from the PacI/NotI fragment derived from C13-18 cosmid.

p-0033<figref idrefs="DRAWINGS">FIG. 5</figref> shows the results of sequencing and primer extension reactions carried out in parallel, which demonstrate, within the AgeI/SacI fragment described in <figref idrefs="DRAWINGS">FIG. 1</figref>, the primary site for the initiation of transcription by chicken RNA polymerase I.

p-0034<figref idrefs="DRAWINGS">FIG. 6</figref> is a diagram that shows the CAT levels measured in the cytoplasmic extracts of cells transfected with plasmids coding for pseudo influenza RNA under the control of human or chicken PolI promoters (in ng/10⁶of transfected cells at 24 hours post-transfection).

p-0035The originality of the present invention relates to a polynucleotide sequence which preferably possesses a transcription-promoting activity, principally in avian cells. The inventors identified this promoting sequence as being the chicken RNA polymerase I promoter.

p-0036The invention also relates to the implication of this polynucleotide in the production of sequences of interest, such as the recombinant influenza viruses provided by reverse genetics systems, for example.

p-00371. Polynucleotide and Recombinant DNA

p-0038In a first aspect, the aim of the invention is an isolated or purified polynucleotide that includes sequence SEQ ID NO: 1 presented in <figref idrefs="DRAWINGS">FIG. 1</figref> or sequence SEQ ID NO: 2 presented in <figref idrefs="DRAWINGS">FIG. 2</figref>, or fragments thereof. Preferably, this polynucleotide, or one of its fragments, possesses a transcription-promoting activity.

p-0039With respect to fragments, the fragments which are preferred are those whose sequences include at least 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 75, or 100 consecutive nucleotides of sequence SEQ ID NO: 1 or SEQ ID NO: 2 and which have a transcription-promoting activity.

p-0040Although the polynucleotide and the promoter in accordance with the invention are preferably characterized by the fact that they possess a transcription-promoting activity in avian cells, this promoter particularly possesses the capacity to promote transcription in poultry cells, and even more particularly in chicken cells.

p-0041By nucleotide sequence, nucleic acid, nucleic sequence or nucleic acid sequence, polynucleotide, and polynucleotide sequence, all terms which will be used in the present application, it is intended to indicate a precise sequence of nucleotides, modified or not, which make it possible to define a fragment or a region of a polynucleotide that may or may not contain non-natural nucleotides, and which may correspond as well to a double-stranded DNA or to a single-stranded DNA. This also includes molecules of DNA, molecules of RNA, cDNA, artificial sequences, and all fragments thereof. It goes without saying the definitions “derived”, “variant”, and “mutated” also apply to the polynucleotides in accordance with the present invention. All polynucleotides that have been modified chemically, enzymatically, or metabolically but that preserve the properties of the polynucleotide of origin, which is to say, a transcription-promoting activity in avian cells, are included in the scope of the present invention.

p-0042It must be understood that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in their natural state. The present invention concerns sequences which have been isolated and/or purified, that is they have been taken directly or indirectly, for example by cloning, amplification, and/or chemical synthesis, and whose environment has been at least partially modified. It must be understood that a polynucleotide introduced into an organism by transformation, genetic manipulation, or by any other method of recombination is “isolated” even if it is present in the aforesaid organism.

p-0043The promoting sequences in accordance with the invention preferably possess a sequence of DNA that present a percentage of identity greater than 70% with one or the other of sequences SEQ ID NO: 1 and SEQ ID NO: 2 described in <figref idrefs="DRAWINGS">FIGS. 1 and 2</figref>. More particularly, the promoting sequences in accordance with the invention present a percentage of identity greater than 80%, and even more preferably 90%, with one or the other of sequences SEQ ID NO: 1 and SEQ ID NO: 2 described in <figref idrefs="DRAWINGS">FIGS. 1 and 2</figref>, or fragments thereof. By “percentage of identity” it is meant a percentage which indicates the degree of identity between two sequences of nucleic acids along the sequences in their entirety. If the sequences considered are of different sizes, the percentage of identity is expressed as a function of the total length of the shortest sequence. To calculate the percentage of identity, the two sequences are superimposed in order to maximize the number of identical bases by permitting intervals, and then the number of identical bases is divided by the total number of bases of the shortest sequence.

p-0044Another aspect of the invention relates to a recombinant DNA that includes a polynucleotide such as defined above.

p-0045This recombinant DNA may contain, for example, the promoting sequence SEQ ID NO: 1 presented in <figref idrefs="DRAWINGS">FIG. 1</figref> or sequence SEQ ID NO: 2 presented in <figref idrefs="DRAWINGS">FIG. 2</figref>, or derivatives thereof, in. which a cloning site is inserted to thus facilitate the use of this sequence as a “portable” promoter. In accordance with a preferred method, the recombinant DNA of the present invention contains, moreover, a sequence to be transcribed. By “sequence to be transcribed” or “sequence of interest”, it is meant a sequence capable of being used as matrix for the synthesis of RNA molecules, such as vRNA. Preferably, the sequence to be transcribed is a cDNA of an uncapped RNA virus, which is to say that the ends of the transcription product should not contain additional nucleotides. More preferably, the sequence to be transcribed is a cDNA of a negative-polarity RNA virus, such as an orthomyxovirus or a paramyxovirus. As an example, the orthomyxovirus could be an influenza, preferably Type A, while the paramyxovirus could be chosen preferably from among the genus <i>Rubulavirus </i>(preferably the mumps virus or the Newcastle's disease virus), the genus <i>Morbillivirus </i>(preferably the measles virus), the genus <i>Pneumovirus </i>(preferably respiratory syncytial virus), or the genus <i>Metapneumovirus</i>. In a preferred method in accordance with the invention, the recombinant DNA is characterized such that it expresses orthomyxovirus vRNA, such as an influenza virus, and preferably a vRNA chosen from among influenza virus vRNA segments 1 to 8. The “sequence to be transcribed” or the “sequence of interest” can also be useful for the production of antisense RNA, which is to say that the structure ensures, by hybridization with the target sequence (a negative-polarity RNA virus, for example), inhibition of the replication or transcription of viral RNA or inhibition of the expression of a corresponding protein product.

p-0046The present invention also relates to vectors that contain the recombinant DNA previously described. These vectors allow the introduction and perhaps the expression of the sequence to be transcribed in a host cell. As examples of vectors, those which may be cited include plasmid expression vectors, viral vectors, integrative vectors, and autosomal vectors. More particularly, a vector in accordance with the present invention is the plasmid pGEM-ChPolI-C15. This plasmid is contained in the strain pGEM-ChPolI-C15-<i>E. Coli </i>1305 deposited, in accordance with the Treaty of Budapest, to the French national microorganism culture collection CNCM (<i>Collection Nationale de Cultures de Micro</i>-<i>organismes</i>), Pasteur Institute, 28, rue de Dr. Roux, 75724 PARIS (France) on Feb. 24, 2003 under the number I-2976. The plasmid pGEM-ChPolI-C15 is derived from the plasmid pGEM52F+ (Promega). A 524 bp EaeI restriction fragment derived from the sequence coding for bacterial chloramphenicol acetyl transferase (CAT) was cloned into the NotI site of pGEM52F+. An AgeI-SacI restriction fragment derived from a C13-18 cosmid was cloned into the pGEM5F+ EcoRI site, upstream from the CAT insert. This 1260 bp fragment contains the minimal chicken RNA polymerase I promoter of the present invention.

p-0047The invention also relates to the production of transgenic birds that express, under the control of the PolI promoter, RNA that confer resistance to infections by pathogenic microorganisms, in particular resistance to avian influenza viruses.

p-00482. Cells and Fertilized Eggs of Avian Origin

p-0049In another aspect, the invention relates to cells transformed by a polynucleotide, and/or a recombinant DNA, and/or a vector as previously described. Preferably, the host cell thus transformed is of avian origin. In accordance with a preferred variation of the invention, the host cell is a poultry cell, and even more preferably a chicken cell. It is understood that in the sense of the present invention the host cells are transformed in a steady manner. However, it is possible to provide transformed cells in a transitory manner.

p-0050In a related aspect, the present invention also relates to “transfected” fertilized eggs of avian origin (preferably from hens). More particularly, the fertilized eggs of the present invention include a polynucleotide, and/or a recombinant DNA, and/or a vector as previously described.

p-0051The methods employed to introduce, into a host cell or fertilized egg, a polynucleotide, and/or a recombinant DNA, and/or a vector as previously described, in accordance with the present invention, is based on the general knowledge of a professional in the fields of cellular transfection and egg incubation, and thus these methods will not be described in greater detail.

p-00523. Methods and Procedures

p-0053In accordance with another aspect, the invention relates to the production of sequences of interest such as, for example, recombinant vaccine viruses, and more particularly to the production of an uncapped recombinant RNA virus. In an additional aspect, the invention targets the use of at least one of the following elements for the production of a sequence of interest, of an uncapped recombinant RNA virus, for example: <ul><li id="ul0007-0001" num="0000"><ul><li id="ul0008-0001" num="0064">a polynucleotide in accordance with the invention;</li><li id="ul0008-0002" num="0065">a recombinant DNA in accordance with the invention;</li><li id="ul0008-0003" num="0066">a vector in accordance with the invention;</li><li id="ul0008-0004" num="0067">a host cell in accordance with the invention; and</li><li id="ul0008-0005" num="0068">a fertilized egg of avian origin in accordance with the invention.</li></ul></li></ul>

p-0054In a related aspect, the present invention proposes a method for the production of uncapped recombinant viruses. Such production is based on a reverse genetics system of recombinant virus production as previously described. More particularly, the method in accordance with the present invention comprises the following steps: <ul><li id="ul0009-0001" num="0000"><ul><li id="ul0010-0001" num="0070">a) introducing into a cell or fertilized egg of avian origin one or several vectors, the aforesaid vector or vectors comprising a recombinant DNA in accordance with the invention, and the DNA that express the proteins necessary for transcription/replication viral RNA (such as proteins PA, PB1, PB2, and NP of an influenza virus, for example);</li><li id="ul0010-0002" num="0071">b) applying to the cell or egg obtained in a) conditions that make possible the expression of recombinant viruses; and</li><li id="ul0010-0003" num="0072">c) recovering the recombinant viruses obtained in b).</li></ul></li></ul>

p-0055It will be understood that in step a) all DNA (recombinant DNA in accordance with the invention, and the DNA that express the proteins necessary to transcribe/replicate viral RNA) can be carried by a single vector, or two vectors, or more.

p-0056In a preferred embodiment, the present invention also proposes a method for the production of recombinant influenza viruses which comprises the following steps: <ul><li id="ul0011-0001" num="0000"><ul><li id="ul0012-0001" num="0075">a) introducing into an avian cell or fertilized chicken egg at least one vector comprising preferentially a recombinant DNA that expresses an influenza virus vRNA and vectors that express proteins PA, PB1, PB2, and NP of an influenza virus such as, preferably, Type A influenza;</li><li id="ul0012-0002" num="0076">b) applying to the cell or egg obtained in a) conditions that make possible the expression of recombinant influenza viruses; and</li><li id="ul0012-0003" num="0077">c) recovering the recombinant influenza viruses obtained in b).</li></ul></li></ul>

p-0057It will be understood that step c) of the methods in accordance with the invention, which comprises the recovery of the recombinant viruses, may take place directly in the cells, after cell lysis, or perhaps from the supernatant. If fertilized eggs are used, a person skilled in the art will know which methods to use in order to recover the recombinant viruses thus produced.

p-00584. Composition

p-0059The present invention also relates to the use of these polynucleotides and recombinant DNA, and/or vectors that contain them, for the preparation of a composition to be used as vaccines. More specifically, a composition in accordance with the present invention includes, moreover, a sequence transcribed by a previously described polynucleotide. In a preferred embodiment, the present invention relates to the use of recombinant viruses for the preparation of vaccine compositions.

p-0060In another preferred embodiment, the composition of the present invention contains, moreover, a pharmaceutically acceptable vehicle and a recombinant virus obtained by a production method in accordance with the present invention.

p-0061The compositions in accordance with the present invention may take on the typical solid or liquid forms used for pharmaceutical administration, which is to say, for example, forms of administration including liquid, gel, or any other medium that permits, for example, controlled release. Among the compositions which may be used, of particular note are the injectable compositions specifically intended for injections into the human circulatory system.

p-0062A person skilled in the art will be able to prepare pharmaceutically acceptable compositions and to determine, as a function of several factors, the favored method of administration and the quantity that must be administered. The factors that may influence this choice include: the exact nature of the active and inactive ingredients included in the composition, the type of viral illness, the patient's condition, age, and weight, etc.

p-0063The following examples will demonstrate other characteristics and advantages of the present invention.

EXAMPLES

p-0064The examples which follow serve to illustrate the extent of the use of the present invention and not to limit its scope. Modifications and variations can be undertaken without escaping the spirit and scope of the invention. Although other methods and products equivalent to those found below can be used to test or carry out the present invention, the preferred materials and methods are described.

p-0065The reverse genetics systems used to obtain recombinant influenza viruses from cDNA clones are based on the transfection of plasmids that code for viral RNA (vRNA) under the control of the human RNA polymerase I promoter (Neumann et al., PNAS 96:9345-50, 1999; Fodor et al., J. Virol. 73:9679-82, 1999; Hoffman et al., PNAS 97:6108-13, 2000). The use of this promoter is justified by the fact that the ends of the transcription products should not contain additional nucleotides (if this is the case, the transcription/replication signals located at the non-coding 5′ and 3′ ends of the vRNA will no longer function).

p-0066The use of the human RNA polymerase I promoter ensures that transcription is initiated at a precise site, and thus ensures the precision of the 5′ end of the vRNA synthesized in the transfected cells. But because of the very narrow species-specificity of RNA polymerase I promoters (Heix and Grummt, Curr. Op. Gent. Dev. 5:652-56, 1995), this type of system can be applied only to cells of human or simian origin.

p-0067However, the availability of a reverse genetics system appropriate for avian cells could open the way for the production of recombinant influenza vaccines in fertilized chicken eggs, which are the only medium currently authorized for the production of influenza vaccine in the United States. Today, vaccination constitutes the single means of influenza prevention, and it is strongly recommended for certain categories of subjects at risk. Production of the vaccine is based on the reproduction of vaccine strains in fertilized eggs, and requires with each new formulation the selection of reassortant viruses that have HA and NA segments representative of the strains in circulation, with the other segments being derived from a donor strain adapted to growth in eggs. It may be of advantage to replace this long and uncertain process with the production of virus reassortants by reverse genetics in fertilized eggs.

p-0068Thus, from the perspective of developing a recombinant influenza virus production system by reverse genetics in avian cells, the inventors undertook to clone the chicken RNA polymerase I promoter. Cloning by PCR based on the homologies between nucleotide sequences of the PolI promoters cloned to date was not possible because PolI transcription systems are widely divergent from one species to another. Thus, the inventors proceeded to screen the chicken genome.

h-00071) Obtaining Cosmid C13-18

p-0069The mapping of the chicken genome indicated that the genes coding for ribosomal RNA (NOR locus) were located on chromosome 16, near the locus of the histocompatibility complex genes (Schmid et al., Cytogenet. Cell Genet., 90:169-218, 2000). In 1988, the laboratory of Professor H. Auffray, part of whose work at the Gustave Roussy Institute in Villejuif involves the major histocompatibility complex, published data concerning a recombinant cosmid (C13-18) which contained 30 kb of chicken genomic DNA, including major histocompatibility complex genes, but also several transcription units of the NOR locus and, consequently, at least one copy of the PolI promoter (Guillemot et al., EMBO J., 7:2775-85, 1988). This cosmid was graciously provided by R. Zoorob.

h-00082) Southern Blot Analysis of C13-18 Cosmid

p-0070The C13-18 cosmid was analyzed by Southern blot after digestion by restriction enzymes NotI, PacI and SfiI, separately and in combination, and using as probes oligonucleotides chosen on the basis of the partial sequencing data for chicken ribosomal RNA available from the databanks:

p-0071<tables id="TABLE-US-00001" num="00001"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="154pt" align="left" /><colspec colname="2" colwidth="63pt" align="left" /><colspec colname="3" colwidth="0pt" align="left" /><tbody valign="top"><row><entry>oligonucleotide 28S/+/2:</entry><entry /><entry /></row><row><entry>5′-GAGTCAGCCCTCGACACAAGCTTTTG-3′;</entry><entry>(SEQ ID NO: 3)</entry></row><row><entry /></row><row><entry>oligonucleotide 18S/−/1.5:</entry></row><row><entry>5′-CTACTGGCAGGATCAACCAGGT-3′;</entry><entry>(SEQ ID NO: 4)</entry></row><row><entry>and</entry></row><row><entry /></row><row><entry>oligonucleotide 18S/−/4:</entry></row><row><entry>5′-TAGAGGAGAACGCGACCTCGAGAC-3′.</entry><entry>(SEQ ID NO: 5)</entry></row></tbody></tgroup></table></tables>

p-0072This analysis made it possible to construct a rough restriction map of the C13-18 cosmid and to position the 18S and 28S ribosomal RNA with respect to the variety of restriction sites located on the cosmid. It also led to the identification of an approximately 16 kb PacI-NotI fragment, corresponding to the majority of an intergenic region and, by consequence, highly likely to contain a copy of the PolI promoter sequence.

h-00093) Cloning the PacI-NotI Restriction Fragment Into a “Reporter” Vector and Demonstrating the Presence of a Transcriptional Promoter Within the PacI-NotI Fragment

p-0073In order to test for the presence of the PolI promoter sequence within the PacI-NotI restriction fragment, the inventors sought to clone this restriction fragment upstream of a reporter sequence.

p-0074For this purpose, the pTCF cosmid (Grosveld et al., 1982, Nucleic Acids Res., 10, 6715-32), which has a single SalI cloning site, was modified by inserting into the SalI site a synthetic sequence that contained multiple cloning sites (StuI-PacI-NotI-PmeI).

p-0075The 16 kb PacI-NotI fragment derived from C13-18 was cloned into the modified pTCF cosmid, at the newly introduced PacI and NotI sites. Two pTCF-(PacI-NotI) recombinant cosmid clones were selected (nos. 2.5 and 2.7). Then a 500 bp EaeI restriction fragment derived from the bacterial chloramphenicol acetyl transferase (CAT) gene was cloned into the NotI site on the pTCF-(PacI-NotI) cosmids, in one orientation and the other. Four recombinant cosmid clones were selected: no. 2.5.3 and no. 2.7.13 (CAT in+orientation), no. 2.5.4 and no. 2.7.16 (CAT in−orientation).

p-0076In order to test for the presence of the PolI promoter sequence within the PacI-NotI restriction fragment, cells from the QT6 quail fibrosarcoma line were transfected with pTCF-(PacI-NotI)-CAT(+) and pTCF-(PacI-NotI)-CAT(−) cosmids. RNA was extracted from the cells 24 hours after transfection using the reagent TriZol (Gibco-BRL) and then treated with DNase (RNase-free DNase, Ambion) in order to eliminate the cosmid molecules that can contaminate RNA preparations. RNAs were deposited at 5 μg per point on a nylon membrane (Hybond N+, Amersham), and hybridized with a ³²P-labeled riboprobe corresponding to the CAT (+) sequence. Positive signals were obtained with the RNA prepared from cells transfected with pTCF-(PacI-NotI)-CAT(−) cosmids nos. 2.5.4 and 2.7.16, which suggests the presence of a transcriptional promoter in the PacI-NotI restriction fragment. In these first experiments, nevertheless, the background signal-to-noise ratio was rather low. It was improved in the following experiments after optimizing the RNA treatment. conditions in DNase (twice for 1 hour at 37° C. in the presence of 8 units of enzyme) and the hybridization and membrane washing conditions (hybridization overnight at 65° C. in a buffer of 50% formamide, 5×SSC, 5× Denhart's, 0.5% SDS; 2 washings of 10 min. at ambient temperature in a buffer of 2×SSC, 0.1% SDS, followed by 2 washings of 10 min. at 65° C. in a buffer of 0.2×SSC, 0.1% SDS). The methodology, which was then applied in an iterative manner to examine the presence of a transcriptional promoter in the restriction fragments derived from the PacI-NotI fragment, is summarized in <figref idrefs="DRAWINGS">FIG. 3</figref>. The results obtained are detailed below and are summarized in <figref idrefs="DRAWINGS">FIG. 4</figref>.

h-00104) Creating Partial Deletions within the Cloned PacI-NotI Restriction Fragment of the Reporter Vector and Demonstrating the Presence of a Transcriptional Promoter in an Approximately 6 kb SfiI-NotI Fragment

p-0077In order to identify more precisely the region of the PacI-NotI fragment that contained the promoter, partial deletions of the Pacd-NotI fragment were obtained by digesting cosmids pTCF-(PacI-NotI)-CAT(−) no. 2.7.16 and pTCF-(PacI-NotI)-CAT(+) no. 2.7.13 with the enzyme combinations PacI-SfiI and SfiI-NotI (the SfiI site being located within the PacI-NotI fragment), filling-in the ends using the Kleenow sub-unit of <i>E. coli </i>PolI DNA, and religating the molecules obtained to themselves. The presence of a transcriptional promoter within the deleted cosmids was tested using the methodology described above (transfection of QT6 cells and analysis of the RNA extracted from the transfected cells by hybridization with a CAT probe). The results obtained suggested that a transcriptional promoter was present in the cosmids that had retained the SfiI-NotI portion (approximately 6 kb) of the Pacd-NotI fragment.

h-00115) Iterative Analysis of the Restriction Fragments Derived From the SfI-NotI Fragment and Demonstrating the Presence of a Transcriptional Promoter in a 1200 bp AgeI-SacI Fragment

p-0078In order to continue the analysis of restriction fragments derived from the SfiI-NotI fragment following the same methodology, a new reporter vector was constructed at this stage. Indeed, the restriction fragments analyzed were still of sufficiently reduced size to be able to be cloned into a plasmid.

p-0079This new reporter vector was constructed from the plasmid pGEM-5-zf+ (Promega), in which the 500 bp EaeI fragment derived from the bacterial CAT gene was cloned in orientation (+) at the single NotI site. Only this orientation of the CAT sequence in the reporter vector was used because the preceding experiments had indicated that the CAT (+) reporter sequence/CAT (−) riboprobe pair gave results that were more specific and reproducible than the CAT (−) reporter sequence/CAT (+) riboprobe pair.

p-0080The SfiI-SacI restriction fragments (in practice, a StuI-SacI fragment that includes the SfiI-SacI fragment) and the SacI-NotI restriction fragments derived from the SfiI-NotI fragment were subcloned into the EcoRV site of the pGEM-CAT (+) vector (clone no. 9), which is located upstream of the CAT reporter sequence. By the methodology described above, the presence of a transcriptional promoter was demonstrated in the StuI-SacI fragment cloned into the plasmid pGEM-(StuI-SacI)-CAT(+) (clone no. 12).

p-0081A restriction map of the StuI-SacI fragment was established based on pGEM-(StuI-SacI)-CAT(+) no. 12 plasmid digestions by SmaI and AgeI restriction enzymes. Two SmaI-SmaI restriction fragments, an AgeI-AgeI restriction fragment, and the StuI-SacI fragment deleted from this AgeI-AgeI restriction fragment were subcloned into the vector pGEM-CAT(+) no. 9 at the EcoRV site. The presence of a transcriptional promoter was demonstrated in the StuI-SacI fragment deleted from the AgeI-AgeI fragment, cloned into the plasmid known by convenience as V12-AgeI (clone no. 29).

p-0082The deleted StuI-SacI fragment was cut into two fragments, StuI-AgeI and AgeI-SacI, obtained by digesting plasmid V12-AgeI no. 29 with the NcoI+AgeI restriction enzymes on the one hand (for the StuI-AgeI fragment), and AgeI+NotI on the other hand (for the AgeI-SacI fragment). Each of the two fragments was subcloned into the pGEM-CAT(+) vector at the EcoRV site. The presence of a transcriptional promoter was demonstrated in the 1200 bp AgeI-SacI fragment, cloned into the plasmid pGEM-(AgeI-SacI)-CAT(+) (clone no. 15).

h-00126) Sequencing the AgeI-SacI Fragment and Determining the Transcription Initiation Point by the Primer Extension Technique

p-0083The AgeI-SacI fragment cloned into the plasmid pGEM-(AgeI-SacI)-CAT(+) no. 15 was sequenced. The sequence is represented in <figref idrefs="DRAWINGS">FIG. 1</figref> (SEQ ID NO: 1).

p-0084The 800 bp distal to this sequence contain 8 repetitions of a sequence of approximately 90 nucleotides, which is characteristic of PolI promoters: indeed, the presence of repeated activating sequences upstream of the transcription initiation site was described for several PolI promoters derived from other species (Paule M., 1998, Promoter Structure of Class I Genes. In <i>Transcription of Ribosomal RNA Genes by Eukaryotic RNA Polymerase I</i>. Edited by M. R. Paule: Springer-Verlag and R.G. Landes Company).

p-0085The precise site of chicken PolI promoter transcription initiation was thus sought in the 400 bp proximal to the AgeI-SacI fragment, by the primer extension technique. An oligonucleotide complementary to the CAT sequence transcribed from the plasmid pGEM-AgeI-SacI)-CAT(+) (sequence SEQ ID NO: 6: 5′-ATGTTCTTTACGATGCGATTGGG-3′) was labeled with ³²P at its 5′ end using T4 phage polynucleotide kinase (Biolabs) and used in two parallel reactions: 1) as primed for the extension of a DNA complementary to the CAT RNA extracted from QT6 cells transfected with the plasmid pGEM-(AgeI-SacI)-CAT(+) no. 15 in the presence of reverse transcriptase (SuperScript II, Invitrogen), and 2) as primed for the sequencing of the plasmid pGEM-(AgeI-SacI)-CAT(+) no. 15 using a “T7 Sequencing Kit” (Pharmacia Biotech). A single transcription product was detected in the primer extension reaction, confirming the existence of a very precise transcription initiation site. This site was located in a region rich in A and T, in agreement with the data available for PolI promoters derived from other species, approximately 200 bp from the oligonucleotide used as a primer.

p-0086In parallel, the analysis of restriction fragments derived from the AgeI-SacI fragment was pursued in order to delimit the minimal promoter (which corresponds to a region of approximately 250 bp for the PolI promoters of other species) (see <figref idrefs="DRAWINGS">FIG. 5</figref>). The AgeI-SacI fragment was cut into three fragments, AgeI-BsaI, BsaI-BsaI, and BsaI-SacI, obtained by digesting the plasmid pGEM-(AgeI-SacI)-CAT(+) no. 15 with restriction enzymes NcoI+BsaI on the one hand (for the AgeI-BsaI and BsaI-BsaI fragments), and BsaI+NotI on the other hand (for the BsaI-SacI fragment). Each of the two fragments was subcloned into the pGEM-CAT(+) vector at the EcoRV site. Only the approximately 600 bp BsaI-SacI fragment (<figref idrefs="DRAWINGS">FIG. 2</figref>), cloned into the plasmid pGEM-(BsaI-SacI)-CAT(+) (clone no. 16), allowed the transcription of the CAT reporter sequence, which is perfectly consistent with the fact that it was the proximal fragment in which the transcription initiation site was located.

p-0087The nucleotide +1 to the transcription initiation site was determined by the primer extension technique, using an oligonucleotide located about fifty base pairs downstream from the initiation region (see <figref idrefs="DRAWINGS">FIG. 5</figref>). This oligonucleotide of sequence SEQ ID NO: 7 (5′-GGCCGGTCAACCCTGCTC-3′) was labeled with ³²P at its 5′ end using T4 phage polynucleotide kinase (Biolabs) and was used in two parallel reactions: 1) as primed for the extension of a DNA complementary to the CAT RNA extracted from QT6 cells transfected with the plasmid pGEM-(AgeI-SacI)-CAT(+) no. 15 in the presence of reverse transcriptase (SuperScript II, Invitrogen), and 2) as primed for the sequencing of the plasmid pGEM-(AgeI-SacI)-CAT(+) no. 15 using a “T7 Sequencing Kit” (Pharmacia Biotech). A majority transcription product was detected, corresponding to an initiation site that possesses a characteristic nucleotide environment (nucleotides underlined below) of the consensus sequence established from the PolI promoter sequences of other species:

p-0088<tables id="TABLE-US-00002" num="00002"><table frame="none" colsep="0" rowsep="0" pgwide="1" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="259pt" align="left" /><colspec colname="2" colwidth="63pt" align="left" /><colspec colname="3" colwidth="119pt" align="left" /><tbody valign="top"><row><entry>                      +1</entry><entry /><entry /></row><row><entry>TTATATGTTCGTC<u> T</u> G <u>T</u>* <u>A</u> G G A G C G A <u>G</u> T G A G* G* ACTCGGCT.</entry><entry>(SEQ ID NO: 8)</entry></row></tbody></tgroup></table></tables>

p-0089It should be noted, however, that three other minority transcription product types were detected in this primer extension experiment, corresponding to initiations at nucleotides −1, +13, and +14 (nucleotides above marked with an asterisk).

p-0090Plasmids intended to express a pseudo-influenza RNA carrying the CAT gene reporter sequence under the control of the chicken PolI promoter were constructed on the model of plasmid pPolI-CAT-Rz designed by Pleschka et al. using human PolI promoter (1996, J. Virol., 70:4188-92): these plasmids (pPR7-C16-CAT-Rz and pPR7-ΔC16-CAT-Rz) contain the sequences coding for the CAT gene, cloned in the anti-sense, and flanked by non-coding 5′ and 3′ sequences derived from the NS gene segment of a human Type A influenza virus; positioning the non-coding 5′ end downstream from nucleotide −1 of the chicken PolI promoter, on the one hand, and inserting the hepatitis δ virus ribozyme sequence at the 3′ non-coding end, on the other hand, are intended to ensure the precision of the ends of the transcription product. The plasmid pPR7-C16-CAT-Rz contains nucleotides −1 to −425 of the chicken PolI promoter, whereas the plasmid pPR7-ΔC16-CAT-Rz contains only nucleotides −1 to −246 (for other species, the minimal size of the PolI promoter was estimated at approximately 250 nucleotides).

p-0091The plasmids PolI-CAT-Rz (human PolI promoter), pPR7-C16-CAT-Rz, and pPR7-ΔC16-CAT-Rz (avian PolI promoter) were transfected into avian QT6 cells and primate COS-1 cells simultaneously with plasmids coding for influenza virus proteins PB1, PB2, PA, and NP. If the transfected cells simultaneously express the correct pseudo-influenza RNA at the non-coding 5′ and 3′ ends and proteins PB1, PB2, PA and NP, the pseudo-influenza RNA can be transcribed and replicated, and the efficacy of the transcription/replication process can be estimated by measuring the level of CAT protein in the transfected cells. Thus, CAT protein levels in cytoplasmic extracts from the transfected cells were measured by ELISA, prepared 24 hours post-transfection.

p-0092The CAT levels measured in the presence of the plasmid pPolI-CAT-Rz were on the order of 200 ng/10⁶transfected COS-1 cells, whereas they were very low (&lt;0.06 ng/10⁶transfected cells) with respect to the QT6 cells (<figref idrefs="DRAWINGS">FIG. 6</figref>).

p-0093On the contrary, the CAT levels measured in the presence of plasmids pPR7-C16-CAT-Rz and pPR7-ΔC16-CAT-Rz were very low (&lt;0.06 ng/10⁶transfected cells) in COS-1 cells, but the levels were on the order of 200 and 100 ng/10⁶cells, respectively, in QT6 cells (<figref idrefs="DRAWINGS">FIG. 6</figref>).

p-0094These results establish that the chicken PolI promoter sequences cloned into plasmids pPR7-C16-CAT-Rz and pPR7-ΔC16-CAT-Rz allow, after transfection into cells of avian origin, the transcription in vivo of mini-replicons that contain influenza virus Type A replication signals. Thus, they open the way for the development of reverse genetics systems that make it possible to obtain recombinant influenza viruses from avian cells in culture, or directly in fertilized chicken eggs.